1. Field of the Invention
The present invention relates to reference libraries of composite spectra that consolidate, into a single searchable data set, information from multiple independent spectra of a chemical compound taken under multiple conditions. The reference libraries may be used, for example, to increase the analytical power of mass spectrometers such as API-CID mass spectrometers.
2. Description of Related Art
A mass spectrometer (MS) is one of the most powerful tools available for quantitative and qualitative analysis of an unknown or suspect compound. One type of mass spectrometer, available since the 1950's, employs electron impact (EI) ionization and fragmentation—whereby vaporized molecules are simultaneously ionized and fragmented with high energy under high vacuum to produce a broad spectrum of ions. EI-MS is typically coupled to a gas chromatograph (GC) to insure adequate molecule separation prior to analysis. Spectra obtained by this technique provide unique spectral “fingerprints” of molecules that are reproducible under set conditions, e.g., 70 eV electron energy and approximately unit resolution.
With the increasing power and availability of computers, large collections, or libraries, of EI-MS spectra have been developed along with associated searching software. Such databases, which may contain hundreds of thousands of molecular fingerprints, are often purchased with the instrument. A typical library search compares the spectrum of an unknown compound with the spectra of known compounds in the library and retrieves “hits” of compounds that have similar spectra.
Unfortunately, GC separation, which is the separation technique usually employed with EI-MS, is not possible with a significant percentage of molecules. GC separation requires that molecules be injected as a vapor. Thus, large, thermally labile molecules are not always suitable for GC analysis. In addition, GC analysis, as often as not, requires extensive and time consuming sample preparation and derivatization. This is especially true in the case of compounds with high polarity and low volatility.
In contrast, liquid chromatography (LC) is a more versatile separation technique. For LC separation, the molecules are injected in solution. Most molecules of interest are more easily solubilized than vaporized. However, LC is not compatible with EI, where the molecule must be introduced into high vacuum as a vapor. Therefore, LC is coupled to mass spectrometers that utilize different mechanisms for ionization, e.g., atmospheric pressure ionization (API).
LC-API-MS is well suited to the investigation of semi-volatile, thermo-labile and polar substances, like pesticides, explosives and forensically relevant substances. Unfortunately, existing El-MS libraries are not suitable for identifying API-MS spectra. New API-MS libraries must be constructed.
One difficulty in constructing libraries for API-MS is the fact that API, unlike EI, generates little or no fragmentation. It is not uncommon to see only the pseudo molecular ion in the mass spectra. This means that API-MS is an excellent means for identifying the molecular weight of an unknown compound, but it cannot distinguish between the thousands of molecules that have any given molecular weight.
To increase ion fragmentation, collision induced dissociation (CID) is typically used in combination with API-MS. However, CID is highly variable. The ions generated by CID, as well as the ion ratios, can vary between different mass spectrometer models, between instruments of the same model, and between day to day operations on the same instrument These variations occur, among other things, as a function of the ion source and the highly sensitive nature of the CID region.
One means for addressing the variable nature of API-CID-MS spectra is the use of performance based tuning. However, even when API-CIDMS is used in conjunction with performance based tuning, the vast array of molecules still vary considerably in their ability to fragment under any given conditions. Therefore, no single set of conditions permits the generation of adequate API-CID-MS spectra for every molecule.
To date, there have been two approaches toward generating reproducible API-CID-MS libraries with sufficiently wide applicability. A first approach generates a composite mass spectrum for a molecule that represents either the sum or the average of multiple spectra taken of the molecule at different CID voltages. However, this technique destroys an important piece of information for identifying the molecule, namely, the way its fragmentation changes relative to changing conditions. A second approach obtains three separate mass spectra of a molecule, each obtained at different CID voltages, and compares each spectrum to a separate library. However, this technique requires the user to sort through three, often contradictory, comparison results using relatively unreliable probability indices due to the few data points in each spectrum.